This revised Program Project competitive renewal submission requests support for advanced studies of genetically engineered herpes simplex virus (HSV) as a novel, yet practical approach for treatment of human brain tumors. The interdisciplinary expertise of investigators at the University of Chicago (Drs. Roizman and Weichselbaum) and the University of Alabama at Birmingham (Drs. Whitley, Markert, Gillespie and Parker) will generate molecular biologic data on mutant HSV and translate this to new Phase I clinical trials in human glioblastoma multiforme. This highly collegial and productive group of investigators proposes a team science approach. Project 1- Roizman will optimize methods for facile production of three entirely novel classes of therapeutic HSV: deltagamma134.5 HSV that express constitutively activated MEK (caMEK), IL12 and are re-targeted to unique cell surface receptors (IL13Ralpha2, uPAR, Grp78 or CD133) expressed specifically and at high abundance on glioma cells in situ; a wild-type HSV that is entirely re-targeted to one of these novel receptors; and, re-targeted HSV that can be administered systemically rather than intratumorally. Project 2- Weichselbaum will further define molecular bases for synergistic anti-tumor interaction between HSV and radiation therapy. From their seminal observation that radiation enhances viral replication and spread within malignant gliomas, a deltagamma134.5 expressing caMEK was generated and will be used to determine how activation of specific cellular gene expression profiles govern resistance of glioma cells to deltagamma134.5 HSV. These data will drive design of new viruses that can exploit this synergistic effect. Project 3- Whitley will focus on GPC subpopulations within human gliomas and will determine whether GPC are differentially resistant to our mutant HSV and whether the oncolytic HSV potential can be improved via specific targeting or expression of complementing pathways identified in Project 2 as supporting enhanced virus replication. Novel viruses and treatment-enhancing discoveries will be used by Project 4- Markert in analyses of human glioma tissues from patients who have participated in 3 G207 HSV Phase I trials at UAB and from patients to be enrolled in the M032 (Interleukin-12-expressing) HSV trial. Synthesis of these findings will lead to a rational design of the best HSV candidate and treatment strategy to advance to the next clinical trial. Three cores support these projects: Administrative- Whitley (including biostatistical support), Experimental Animal Glioma Model- Gillespie (testing safety and efficacy in relevant animal models) and Virus Production- Parker (production/characterization of highly purified, high-titered virus stocks).